Bottom Line:
Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements.The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments.Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]).

ABSTRACTThe Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.

Mentions:
We proceeded to clone the PLOD-1 promoter in a luciferase reporter gene vector. We characterized the response to PITX2 by cotransfecting the PLOD-1–luciferase construct with a PITX2 expression vector. In CHO cells, the PLOD-1 261 construct containing 10 bicoid elements was activated 20-fold by PITX2A (Fig. 4 A). The minimal promoter constructs, PLOD-1 2561, were modestly activated (approximately fivefold) by PITX2. We attribute the modest activation to the fact that two bicoid-like sites remain, one TAACCC and one TAAGCC, in the minimal construct. Such elements are known to bind PITX2 in vitro (Amendt, B., unpublished data). In HeLa cells, PLOD-1 was activated 6.5-fold by PITX2A (Fig. 4 B). Furthermore, we found that a PITX2 gene carrying the Rieger syndrome causing mutation T68P failed to upregulate PLOD-1 in cell culture (Fig. 5). This protein can be expressed in cell culture (Amendt et al. 1998). The responses were similar to the negative controls both in CHO cells (Fig. 5 B) and in HeLa cells (Fig. 5 C).

Mentions:
We proceeded to clone the PLOD-1 promoter in a luciferase reporter gene vector. We characterized the response to PITX2 by cotransfecting the PLOD-1–luciferase construct with a PITX2 expression vector. In CHO cells, the PLOD-1 261 construct containing 10 bicoid elements was activated 20-fold by PITX2A (Fig. 4 A). The minimal promoter constructs, PLOD-1 2561, were modestly activated (approximately fivefold) by PITX2. We attribute the modest activation to the fact that two bicoid-like sites remain, one TAACCC and one TAAGCC, in the minimal construct. Such elements are known to bind PITX2 in vitro (Amendt, B., unpublished data). In HeLa cells, PLOD-1 was activated 6.5-fold by PITX2A (Fig. 4 B). Furthermore, we found that a PITX2 gene carrying the Rieger syndrome causing mutation T68P failed to upregulate PLOD-1 in cell culture (Fig. 5). This protein can be expressed in cell culture (Amendt et al. 1998). The responses were similar to the negative controls both in CHO cells (Fig. 5 B) and in HeLa cells (Fig. 5 C).

Bottom Line:
Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements.The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments.Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]).

ABSTRACTThe Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.